This invention relates to the field of preserving cells. More specifically, it relates to methods of drying and stabilizing prokaryotic cells and the compositions obtained thereby.
Live prokaryotic cells, particularly bacteria, are widely and increasingly used in important medical, agricultural and industrial applications. Agricultural, or environmental, applications include biopesticides and bioremediation. Medical applications include use of live bacteria in vaccines as well as production of pharmaceutical products and numerous industrial compositions. The use of live bacterial vaccines promises only to increase, given the dramatic rise in biotechnology as well as the intensive research into the treatment of infectious diseases over the past twenty years.
Bacterial cells must be able to be stored for significant periods of time while preserving their viability to be used effectively both in terms of desired results and cost. Storage viability has proven to be a major difficulty. Methods for preserving live prokaryotic cells suffer from several serious drawbacks, such as being energy-intensive and requiring cold storage. Furthermore, existing preservation methods fail to provide satisfactory viability upon storage, especially if cells are stored at ambient or higher temperature.
Freeze-drying is often used for preservation and storage of prokaryotic cells. However, it has the undesirable characteristics of significantly reducing viability as well as being time- and energy-intensive and thus expensive. Freeze-drying involves placing the cells in solution, freezing the solution, and exposing the frozen solid to a vacuum under conditions where it remains solid and the water and any other volatile components are removed by sublimation. The resulting dried formulation comprises the prokaryotic cells.
In spite of the apparent ubiquity of freeze-drying, freeze-dried bacteria are unstable at ambient temperatures, thus necessitating storage by refrigeration. Even when refrigerated, however, the cells can quickly lose viability. Damage caused by this process may be circumvented, to a certain degree, by the use of excipients such as lyoprotectants. However, lyoprotectants may subsequently react with the dried cells, imposing inherent instability upon storage of the freeze-dried prokaryotic cells.
Other methods used to prepare dry, purportedly stable preparations of prokaryotic cells such as ambient temperature drying, spray drying, liquid formulations, and freezing of bacterial cultures with cryoprotectants also have drawbacks. For a general review on desiccation tolerance of prokaryotes, see Potts (1994) Micro. Rev. 58:755-805. Ambient temperature drying techniques eliminate the freezing step and associated freeze-damage to the substance, and these techniques are more rapid and energy-efficient in the removal of water. Crowe et al. (1990) Cryobiol. 27:219-231. However, ambient temperature drying often yields unsatisfactory viability. Spray drying results in limited storage time and reduced viability, even when stabilizing excipients are used. For a general review, see Lievense and van""t Reit (1994) Adv. Biochem. Eng. Biotechnol. 51 :45-63; 72-89. Liquid formulations may provide only short-term stabilization and require refrigeration. Freezing bacterial cultures results in substantial damage to the bacterial cell wall and loss of viability which is only reduced but not eliminated by the use of cryoprotectants. Moreover, these frozen cultures also need to be stored refrigerated.
Trehalose, (xcex1-D-glucopyranosyl-xcex1-D-glucopyranoside), is a naturally occurring, non-reducing disaccharide which was initially found to be associated with the prevention of desiccation damage in certain plants and animals which can dry out without damage and can revive when rehydrated. Trehalose has been shown to be useful in preventing denaturation of proteins, viruses and foodstuffs during desiccation. See U.S. Pat. Nos. 4,891,319; 5,149,653; 5,026,566; Blakeley et al. (1990) Lancet 336:854-855; Roser (July 1991) Trends in Food Sci. and Tech. 10:166-169; Colaco et al. (1992) Biotechnol. Internat. 1:345-350; Roser (1991) BioPharm. 4:47-53; Colaco et al. (1992) Bio/Tech. 10:1007-1011; and Roser et al. (May 1993) New Scientist, pp. 25-28. Trehalose dihydrate is available commercially in good manufacturing process (GMP) grade crystalline formulations. A method of making trehalose from starch is described in EP patent publication No. 639 645 A1. This method involves a two step enzymatic bioconversion of starch to yield a trehalose syrup from which the sugar is recovered by crystallisation.
Bacteria are able to counteract osmotic shock by accumulating and/or synthesizing potassium with a few types of organic molecules, including some sugars. Osmoregulation in bacteria such as Escherichia coli in glucose-mineral medium without any osmoprotective compounds involves the endogenous production of trehalose. Larsen et al. (1987) Arch. Microbiol. 147:1-7; Dinnbier et al. (1988) Arch. Microbiol. 150:348-357; Giaever et al. (1988) J. Bacteriol. 170:2841-2849; and Welsh et al. (1991) J. Gen. Microbiol. 137:745-750.
One method of preserving prokaryotic cells is freeze-drying in the presence of trehalose. See, e.g., Israeli et al. (1993) Cryobiol. 30:519-523. However, this method provides unsatisfactory viability. Israeli et al. freeze dried E. coli in the presence of 100 mM trehalose but reported survival data for only four days after exposure of the dried samples to air at 21xc2x0 C. A later study tested survival rates of E. coli and Bacillus fluoringiensis freeze-dried in the presence of trehalose. Leslie et al. (1995) Appl. Env. Microbiol. 61:3592-3597. Survival data were reported only for 4 days after exposure of the dried samples to air.
Another study comparing freeze-dried to air-dried (sealed under nitrogen) E. coli in the presence of trehalose reported survival rates of about 107 to over 1010 colony forming units (CFU) per ml for cells stored for 25 weeks, but the cells were stored at 4xc2x0 C. Louis et al. (1994) Appl. Microbiol. Biotechnol. 41:684-688.
In view of increasing applications for viable bacteria and the existing problems regarding maintaining bacterial viability during storage, there is a pressing need for a method to inexpensively dry and stabilize prokaryotic cells. It is especially desirable to develop methods that would allow storage of dried prokaryotic cells at ambient temperature, i.e., not requiring refrigeration. The methods described herein address this need by providing dry, remarkably storage-stable, prokaryotic cells that retain viability without the need for refrigeration.
All references cited herein are hereby incorporated herein by reference in their entirety.
The present invention encompasses methods of producing dried, stabilized prokaryotic cells. The invention also includes compositions produced by these methods, as well as methods of reconstituting the prokaryotic cells.
A more detailed description of this assay is provided in Example 2. Preferably, residual moisture will be equal to or less than about 5%, more preferably less than about 4%, more preferably equal to or less than about 3% even more preferably equal to or less than about 2.5%. When cells are dried more rapidly by gradually increasing the temperature, as described above, residual moisture may drop below 2%. The allowable maximum for different cell types can easily be determined empirically. Generally, residual moisture above about 5% can be detrimental to viability. This varies depending, inter alia, on the genus/species/strain used, the concentration and type of non-reducing carbohydrate used in the drying solution, method of drying and type of storage.